Relative Ability of Orally Administered Lactobacillus murinus To Predominate and Persist in the Porcine Gastrointestinal Tract

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at ~10¹⁰ CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at ~10⁷ to 10⁸ CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at ~10⁵ CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 (~10⁶ CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted ~10⁷ CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by ~87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.     

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

Paenibacillus larvae Chitin-Degrading Protein PlCBP49 Is a Key Virulence Factor in American Foulbrood of Honey Bees

Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens.     

Significant dreams: Repositioning the self narrative.

In this article the authors argue that the study of the ongoing significance of significant dreams necessarily goes beyond quantitative methods for analyzing dream content to a qualitative study of how the dream experience influences the dreamer's meaning-making processes. A set of concepts from narrative psychology is introduced as being potentially valuable in this regard. A case study is presented to illustrate how the significant dream may serve as a catalyst for repositioning the dreamer's self narrative relative to a cultural master narrative. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Actin mediated release of ATP from a myosin-ATP complex.

The apparent second-order rate constant, ka-2, of actin binding to a myosin-ATP state (M*.ATP) and releasing ATP to the medium has been determined by two methods. The first was the measurement of the amount of ATP released when actin was added to the intermediate state, M*.ATP; the second was the measurement of oxygen exchange between ATP and HOH. A quantitative treatment of ATP in equilibrium HOH exchange is given to allow extraction of elementary rate constants from the data. Agreement between the two methods was good and at low ionic strength and 23 degrees C, ka-2 is 6 X 10(5) M-1 s-1 which is about one-third the value of the apparent second-order rate constant, ka4, of actin binding to the myosin product state (M**.ADP.Pi). The determination of ka-2 allows a lower limit of 6 s-1 to be placed upon the first-order rate of ATP release from AM.ATP. This is to be compared with a value of less than or equal to 1.5 X 10(-4) s-1 for the equivalent steps of the myosin scheme; thus actin enhances the rate by a factor of 4 X 10(4) or more. A greater proportion of the bound ATP is released to the medium as ATP with increasing actin concentration. This reflects the contribution to rate limitation at saturating actin concentration of steps between myosin states dissociated from actin.     

The genome sequence of a reassortant bluetongue virus serotype 3 from India

All 10 genome segments (Seg-1 to 10-a total of 19,188 bp) were sequenced from a strain of bluetongue virus serotype 3 (BTV-3) from India (strain IND2003/08). Sequence comparisons showed that nine of the genome segments from this virus group with other eastern topotype strains. Genome Seg-2 and Seg-6 group with eastern BTV-3 strains from Japan. However, Seg-5 (the NS1 gene) from IND2003/08 belongs to a western lineage, demonstrating that IND2003/08 is a reassortant between eastern and western topotype bluetongue viruses. This confirms that western BTV strains have been imported and are circulating within the subcontinent.     

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.     

Rhizodeposition shapes rhizosphere microbial community structure in organic soil

The aims of the study were to determine group specificity in microbial utilization of root-exudate compounds and whole rhizodeposition; quantify the proportions of carbon acquired by microbial groups from soil organic matter and rhizodeposition, respectively; and assess the importance of root-derived C as a driver of soil microbial community structure. Additions of 13C-labelled root-exudate compounds to organic soil and steady-state labelling of Lolium perenne, coupled to compound-specific isotope ratio mass spectrometry, were used to quantify group-specific microbial utilization of rhizodeposition. Microbial utilization of glucose and fumaric acid was widespread through the microbial community, but glycine was utilized by a narrower range of populations, as indicated by the enrichment of phospholipid fatty acid (PLFA) analysis fractions. In L. perenne rhizospheres, high rates of rhizodeposit utilization by microbial groups showed good correspondence with increased abundance of these groups in the rhizosphere. Although rhizodeposition was not the quantitatively dominant C source for microbes in L. perenne rhizospheres, relative utilization of this C source was an important driver of microbial group abundance in organic soil.